The Effects of Purine Compounds on the Isolated Aorta of the Frog Rana Temporaria Gillian E

The effects of purine compounds on the isolated aorta of the frog Rana temporaria Gillian E. Knight & 'Geoffrey Burnstock

Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT

1 In the isolated aorta of the frog, Rana temporaria, adenosine concentration-dependently, endothelium-independently relaxed adrenaline pre-constricted vessels. None of the adenosine analogues including D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R- and S-PIA) and 2-chloroadenosine (2-CA), or the more selective Al, A2 and A3 agonists cyclopentyladenosine (CPA), CGS 21680 and Ml-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (IB-MECA) respectively, had any effect. 2 The non-selective adenosine antagonist, 8-p-sulphophenyl-theophylline (8-pSPT; 30 giM) failed to inhibit adenosine relaxations, as did NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mM) and indomethacin (30 gM). 3 Adenosine 5'-triphosphate (ATP), ca,f,-methylene ATP (a,#-MeATP), ,B,y-methylene ATP (fl,y- MeATP), 2-methylthio ATP (2-MeSATP) and uridine 5'-triphosphate (UTP) all concentration- dependently contracted the frog aorta. ATP and a,,B-MeATP were equipotent and more potent than UTP and ,B,y-MeATP; 2-MeSATP had little activity. 4 The P2-purinoceptor antagonist, suramin (0.1 mM) inhibited contractions to x,,B-MeATP but not to ATP. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 gM) also inhibited contractions to a,,B-MeATP but not to ATP. Contractions to ATP were, however, inhibited by indomethacin (30 gM). 5 In conclusion, in the frog aorta there appears to be a novel subclass of P1-purinoceptor mediating vasodilatation, although like the A3 subclass it is not blocked by methylxanthines; a P2-purinoceptor mediates vasoconstriction which resembles a P2X subtype, based on the agonist potency of a,,B-MeATP being more potent than 2-MeSATP (UTP has moderate activity) and PPADS is an effective antagonist. There is no evidence for the presence of a P2Y-purinoceptor, mediating vasodilatation, in this preparation. Keywords: Frog aorta; adenosine; ATP; purinoceptor

Introduction Purine nucleotides and nucleosides have long been known to agonist at this receptor (Jacobson et al., 1993; Gallo- have effects on the cardiovascular system of mammals. Ade- Rodriguez et al., 1994) and D-5'-(N-ethylcarbox nosine was shown to inhibit activity in mammalian hearts in amide) adenosine (NECA) and N6-(2-phenylisopropyl) ade- 1929 by Drury & Szent Gy6rgyi, and it was observed that ATP nosine (PIA) are equipotent, but it is resistant to methyl- was more effective at causing heart block in the guinea-pig xanthine blockade. P2-purinoceptors were initially divided into than adenosine (Drury, 1936). Further investigations into the P2x and P2Y subclasses (Burnstock & Kennedy, 1985); other effect of adenosine and ATP on the cardiovascular system subdivisions have since been recognised, P2Z, P2T and P2u followed (Gaddum & Holtz, 1933; Richards, 1934; Green & classes (Gordon, 1986; Hoyle & Burnstock, 199la; Stone, 1991; Stoner, 1950) resulting in detailed documentary evidence of the Hoyle, 1992; O'Connor, 1992). Additional subdivisions have important role of both adenosine and ATP in the maintenance been proposed, P2R, P2S and P3, but these have not been widely of mammalian vascular tone (see Olsson & Pearson, 1990) via accepted (see Hoyle & Burnstock, 1991a, b; Stone, 1991). In the the heart and systemic vascular systems (Su, 1981; 1985; most recent proposal for purinoceptor subclassification by Burnstock & Kennedy, 1986; Burnstock, 1987a,b; 1989; 1990b; Abbracchio & Burnstock (1994), adopted in principle by the Ralevic & Burnstock, 1991). IUPHAR Nomenclature Committee (see Fredholm et al., Once it was recognised that adenosine and ATP had differ- 1994), two families of purinoceptors are recognised, P2X ent effects on mammalian systems and were therefore acting via (ligand-gated cation channels) and P2Y (G-protein mediated), separate receptors, a formal classification was proposed each with their own subclasses, P2XI4 and P2YI-7. (Burnstock, 1978). Receptors selective for adenosine and ade- Both P,- and P2-purinoceptors have important actions on nosine monophosphate (AMP) were designated as PI-pur- the cardiovascular system. Generally, adenosine dilates mam- inoceptors and those selective for ATP and adenosine malian blood vessels by acting on A2-receptors located on the diphosphate (ADP) called P2-purinoceptors. P1-purinoceptors, smooth muscle (Berne et al., 1983; Burnstock & Kennedy, 1986; susceptible to methylxanthine blockade, were divided into Al- Collis, 1989) whereas activation of Al-receptors often mediates and A2-receptors (Van Calker et al., 1979; Londos et al., 1980). prejunctional inhibition (Verhaeghe et al., 1977). Stimulation More recently an A3-receptor subclass has been cloned of P2x-purinoceptors mediates vasoconstriction via an action (Meyerhof et al., 1991; Zhou et al., 1992), M-(3-iodobenzyl)- on receptors located on the smooth muscle (Burnstock & adenosine-5'-N-methylcarboxamide (IB-MECA) is a selective Kennedy, 1986; Burnstock, 1990a, c) and activation of endothelial P2Y-purinoceptors results in vasodilatation, via the release of endothelium-derived relaxing factor (EDRF) now known to be nitric oxide (NO) (De Mey & Vanhoutte, 1981; 'Author for correspondence. Houston et al., 1987; Pearson & Gordon, 1989) and prostacy- 874 G.E. Knight & G. Burnstock Adenosine dilates and ATP constricts the frog aorta clin (Moncada & Vane, 1979), although P2Y-purinoceptors presence of the P2-purinoceptor antagonists, suramin (0.1 mM) have been identified on the vascular smooth muscle of some and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid vessels (Mathieson & Burnstock, 1985; Brizzolara & Burn- (PPADS; 30 gM) and the prostanoid inhibitor, indomethacin stock, 1991). (30 mM). All antagonists were allowed to equilibrate for While the effects of both adenosine and ATP have been 20 min before concentration-response curves were repeated. extensively studied on mammalian systems, there is, in con- trast, only limited information available on the role of purines Drugs used in the control of the cardiovascular system of lower vertebrates. A presynaptic Al-receptor has been identified, stimu- Adrenaline bitartrate, adenosine, NECA, R-PIA, S,PIA, 2- lation of which inhibits sympathetic nerve activity to arterioles CA, ATP, cx,f3-MeATP, f3,y-MeATP, UTP, ACh, in- of frog cutaneous muscle (Fuglsang et al., 1989). There are domethacin, L-NAME, dimethyl sulphoxide (DMSO) and as- several studies examining the effect of purine compounds on corbic acid were supplied by Sigma Chemical Co; 2-MeSATP, the amphibian heart. P1-purinoceptors have been demon- CPA, CGS 21680 (2-[-p-(2-carboxyethyl) phenethylamino]-5'- strated in frog heart (Burnstock & Maghji, 1981; Lazou & N-ethylcarboxamido adenosine) and 8-pSPT by Research Beis, 1987). P2-purinoceptors have been identified in axolotl, Biochemicals Inc.; IB-MECA was the kind gift of Prof. K. frog and toad atria (Meghji & Burnstock, 1983a, b; Hoyle & Jacobson (NIH, U.S.A.), suramin was the kind gift of I.C.I. Burnstock, 1986). A non-adrenergic, non-cholinergic (NANC) and PPADS a kind gift from Prof. Lambrecht (Frankfurt, excitatory transmission has been demonstrated in the frog Germany). Adrenaline was dissolved in 100 gM ascorbic acid; heart (Donald, 1985) which is mediated by a P2-purinoceptor IB-MECA in DMSO to give a stock solution, the first dilution (Burnstock & Meghji, 1981), ATP being a cotransmitter with in 50% DMSO/50% distilled water, and subsequent dilutions adrenaline (Hoyle & Burnstock, 1986; Bramich et al., 1990). in distilled water; indomethacin in 0.2 M NaCO3 solution and Studies have revealed PI-purinoceptors on the coronary artery NECA, R- and S-PIA, CPA and CGS 21680 in DMSO to of trout and skate (Small et al., 1990; Farrell & Davie, 1991) produce a stock solution, with subsequent dilutions in distilled and trout gill vasculature (Colin & Leray, 1979; Colin et al., water. All other drugs were dissolved in distilled water. 1979), dogfish atria and aorta (Meghji & Burnstock, 1984; Evans, 1992). ATP has been shown to dilate the portal vein of Statistical analysis the lizard (Ojewole, 1983). The present investigation was carried out to study the effects of adenosine and ATP in the iso- Vasodilator responses are expressed as mean % relaxation of lated aorta of the frog and to identify purinoceptor subtypes the adrenaline (EC50 concentration) contraction + standard using agonist potency orders and selective antagonists. error (s.e.) (n) and vasoconstrictor responses are expressed as a mean maximum tension developed in g or as a % of the maximum response in the absence of antagonist+s.e. (n). Methods Significance was tested by analysis of variance followed by Tukey's test, or Student's paired t test. A probability of Frogs (Rana temporaria) of either sex, supplied by Blades P < 0.05 was taken as significant. Biological, Cowden, Edenbridge, U.K., were stunned and de- capitated. The frog was pithed and the brain destroyed with a metal seeker. The aortic branches were rapidly removed and Results placed in a physiological saline solution. Ring segments of approximately 4-5 mm, one from each arch, were dissected Vasodilator response to adenosine free and mounted in 10 ml organ baths containing gassed (95% 02/5% C02) saline of the following composition (mM): Adrenaline, known to be the principle transmitter in sympa- NaCl 111.1, KC1 1.88, NaH2PO4 0.08, NaHCO3 2.35, CaCl2 thetic nerves in amphibians (see Burnstock, 1969), concentra- 1.08 and glucose 1.11. All experiments were carried out at tion-dependently constricted isolated preparations of frog room temperature, 23 + 1.00C. aorta. The EC50 value for each preparation was calculated and Segments of aorta were mounted horizontally in the organ this concentration was used to constrict the vessel to study bath by inserting two tungsten wires through the lumen of vasodilator responses. The mean pD2 (-log EC50 value) for the vessel. One wire was used to anchor the vessel to a rigid adrenaline was 6.07 + 0.10 (n = 18). support, the other attached to a Grass force-displacement Adenosine (1 pM-3 mM) concentration-dependently, en- transducer FT03C. Mechanical activity was displayed on a dothelium-independently relaxed adrenaline pre-constricted Grass ink-writing oscillograph. An initial load of 0.75-1 g vessels. As adenosine did not consistently cause a maximum was applied and the vessels were allowed to equilibrate for vasodilatation, a pD2 value could not be calculated. 1 h. The adenosine analogues NECA, R- and S-PIA and 2-CA With each vessel, a concentration-response curve for and the more selective Al, A2 and A3 agonists CPA, CGS adrenaline was constructed in order to obtain an EC50 value. 21680 and IB-MECA respectively, were all without effect, up This concentration of adrenaline was used to constrict the to a concentration of 30 pM (n = 5). vessel to investigate vasodilator responses. The integrity of The non-selective adenosine antagonist 8-pSPT (30 gM) had the endothelium was tested at the beginning of the experi- no effect on the dilator activity of adenosine. Since adenosine ment by the vasodilator response to acetylcholine (ACh; failed to reach a maximum response in either the absence or data not shown). presence of 8-pSPT, pD2 values could not be compared; Vasodilator responses to adenosine and analogues NECA, however, Student's paired t test revealed no statistical differ- R and S-PIA, 2-chloroadenosine (2-CA), cyclopentyladenosine ence in the concentration-response curve for adenosine in the (CPA), CGS 21680 and IB-MECA were examined on adre- absence and presence of 8-pSPT (P > 0.05) (Figure 1). naline (EC5o) pre-constricted vessels. The concentration-re- Concentration-response curves for adenosine were repeated sponse curve to adenosine was repeated in the presence of the in the presence of L-NAME (0.1 mM; n = 6) and indomethacin P1-purinoceptor antagonist 8-p-sulphophenyl-theophylline (8- (30 pM; n = 6). There was no significant difference in con- pSPT, 30 pM), the NO-synthase inhibitor N0-nitro-L-arginine centration-response curves for adenosine in the absence or methyl ester (L-NAME, 0.1 mM) and the prostanoid inhibitor, presence of these agents (P > 0.05). indomethacin (30 pM). Vasoconstrictor concentration-response curves to ATP, a,#- Vasoconstrictor responses to ATP and analogues MeATP, fJ,y-MeATP, UTP and 2-methylthio ATP (2-Me- SATP) were constructed at basal tone. The concentration-re- At basal tone ATP, cxf-MeATP, fi,y-MeATP and UTP all sponse curves to ATP and x,f1-MeATP were repeated in the concentration-dependently constricted the isolated aorta, 2- G.E. Knight & G. Burnstock Adenosine dilates and ATP constricts the frog aorta 875 MeSATP being virtually without activity. None of the analo- Repeated administration of a,f1-MeATP (30 gM; n = 3) did gues reached a maximum response, therefore the purine com- not desensitize the vessel either to an increased concentration pounds were compared by expressing contractions as mean of a,fi-MeATP or to ATP administration and in both cases a tensions developed in mg. Analysis of variance followed by a further contraction was observed. In adrenaline pre-con- Tukey's test revealed an order of potency for the agonists to stricted vessels, neither ATP (up to 3 mM) nor 2-MeSATP (up be: ac,#-MeATP = ATP > UTP = fl,y-MeATP > > 2-MeSATP to 30 gM) caused vasodilatation; ATP generally raised the tone (Figure 2). of the vessel still further, while 2-MeSATP had no effect. Effect of antagonists on constrictor responses 100 Suramin (0.1 mM; n = 6) significantly (P < 0.05) inhibited constrictor responses to a,fl-MeATP (Figure 3a). As maximum 90 responses were not reached, pD2 values could not be calculated. Suramin (0.1 mM; n = 9) had no inhibitory effect on ATP 80 (Figure 3b). PPADS (30 jM) significantly (P <0.05) inhibited con- 70 strictor responses to a,fl-MeATP (n = 6; Figure 4a), but not ATP (n = 5; Figure 4b). C 60 Indomethacin (30 pM) significantly (P< 0.05) inhibited the 0 response to ATP (n = 7; Figure 5), but not ae,f,-MeATP (n = 9). x 50- In the presence of indomethacin (30 gM) neither suramin (0.1 mM; n = 7) nor PPADS (30 ,UM; n = 7) caused any further responses to ATP. K 40 inhibition of the

30- Discussion 20 The vasodilator activity of adenosine in the frog aorta is consistent with its vasodilator action in both mammals (see 10 Burnstock, 1990b) and fish vessels, including the trout and skate coronary arteries (Small et al., 1990; Farrell & Davie, 0 1991) and dogfish aorta (Meghji & Burnstock, 1984). In mammals the subclass of P1-purinoceptor mediating vasodi- 6 5.5 5 4.5 4 3.5 3 2.5 latation of most vessels is the A2-receptor (Burnstock, 1990b). -log (Adenosine) Adenosine dilates the frog aorta in a concentration-dependent, none of the ade- Figure 1 Cumulative concentration-response curve for adenosine on endothelium-independent manner; however adrenaline (EC50 concentration) preconstricted aorta of the frog Rana nosine analogues, which are used to classify the subtype of P1- temporaria in the absence (U, n = 6) and presence (Q, n = 6) of 8- purinoceptor in mammalian systems, had any vasodilator ac- pSPT (30upM). Symbols represent mean % relaxation + s.e. (unless tivity, these include NECA, R- and S-PIA and 2-CA. In ad- masked by the symbol). dition, the selective Al, A2 and A3 analogues CPA, CGS 21680 and IB-MECA respectively (Lohse et al., 1988; Hutchison et al., 1989; Jacobson et al., 1993; Gallo-Rodriguez et al., 1994) were without effect upto a concentration of 30 gM. These more 500 1 selective analogues are significantly more potent than adeno-

450

400 a * b 100- 110- 350 90- 100- 80- 90 - g 300 - E c 0 70- 80- a o 250 - 4L) 60- 70- 0 cJ 50- 60 - 50- w 200 - 0 40- 0 * 40- 110 30- 150- 30- 20- 20 - 10- - 10 100 0- 50 - 7.5 6 5.5 5 4.5 4 3.5 6 5.5 5 4.5 4 3.5 3 2.5 -log (a,P-MeATP) -log (ATP) 0* Figure 3 Concentration-response curves for a,,B-MeATP and ATP in 6 5.5 5- 4.5 4 3.5 3 2.5 the isolated aorta of the frog Rana temporaria. (a) Concentration- -log (Purine) response curve for a,fl-MeATP in the absence (A, n = 6) and presence (A, n = 6) of suramin (0.1 mM). Symbols represent mean % Figure 2 Concentration-response curves for A'TP (0, n = 21) and its contraction+ s.e. (unless masked by the symbol). Indicates statistical stable analogues a,,B-MeATP (A, n = 26), /3,y-N4eATP (O, n = 7) and significance (P <0.05). (b) Concentration-response curve for ATP in 2-MeSATP (*, n = 16) and the pyrimidine U'TP (V, n=8) on the the absence (-, n=9) and presence (0. n=9) of suramin (0.1 mM). isolaed aorta of the frog Rana temporaria. Akll symbols represent Symbols represent mean % contraction + s.e. (unless masked by the mean contraction in mg±s.e. (unless masked tajbv theLAI%,oOjlAAL.fsvmbol). symbol). 876 G.E. Knight & G. Burnstock Adenosine dilates and ATP constricts the frog aorta

a * b 100- 100 100 90- 90 80- 80 90 * a 0 701 * 70 80 C. 60- 60 c(a 50- 50 70 0 40- 40 10I 30- 30 O 60 C, 20- * 20 10- 10 0 O0 0 40 I I .

6.5 6 5.5 5 4.5 4 3.5 5 4.5 4 3.5 3 2.5 -log (aj-MeATP) -log (ATP) 30

Figure 4 Concentration-response curve for a,fi-MeATP and AT? on 20 the isolated aorta of the frog Rana temporaria. (a) Concentration- response curve for o,fi-MeATP in the absence (A, n = 6) and presence 10- (A, n = 6) of PPADS (30 gM). Symbols represent mean % contraction+s.e. (unless masked by the symbol). Indicates statistical significance (P < 0.05). (b) Concentration-response curve for ATP in 0 the absence (0, n=6) and presence (0, n = 6) of PPADS (30mM). Symbols represent mean % contraction+ s.e. (unless masked by the 6 5.5 5 4.5 4 3.5 3 2.5 symbol). -log (ATP)

Figure 5 Concentration-response curve for ATP on the isolated aorta of the frog Rana temporaria in the absence (0, n=7) and presence (0, n = 7) of indomethacin (30 pM). Symbols represent mean sine itself, for instance an IC50 value of 4.3 pM for 2-CA has % contraction + s.e. (unless masked by the symbol). Indicates been reported for the guinea-pig aorta (Alexander et al., 1994) statistical significance (P<0.05). whereas an EC50 of 115 nM for CGS 21680 has been reported in the rat aorta (Conti et al., 1993). CPA modulates ACh release in the mouse hemidiaphragm with an IC50 of 0.08 pM (Nagano et al., 1992). The novel A3 agonist, IB-MECA has a from mammalian P2X-purinoceptors since it is insensitive to Ki value of 1.1 nM in rat brain membranes (Gallo-Rodriguez et a,fl-MeATP desensitization. It is interesting that ATP itself is al., 1994). Further, the non-selective P1-purinoceptor antago- not inhibited by either the nonselective P2-purinoceptor an- nist, 8-pSPT, did not affect the dilator activity of adenosine. tagonist suramin (Dunn & Blakeley, 1988; Hoyle et al., 1990) Thus, the adenosine receptor mediating vasodilatation in the or PPADS. A separate property of suramin is its ability to frog aorta appears to be novel, although it does resemble the inhibit Ca2+/Mg2+-dependent ectoATPases (Hourani & A3-receptor in that methylxanthines are not effective blockers. Chown, 1989; Ziganshin et al., 1995); this fact may account for Adenosine is not initiating relaxation via the production ofNO the lack of antagonism against ATP observed in this pre- or a prostanoid, since neither L-NAME, which inhibits the paration. However, contractions to ATP were inhibited by activity of NO-synthase (Rees et al., 1989; 1990), nor in- indomethacin, demonstrating that much of its action is via the domethacin which inhibits prostanoid production (Vane, production of a prostanoid. UTP also initiates contractions, 1971), had any effect on the dilator response to adenosine. although less potently than either ATP or c,fJ-MeATP. A se- Both NO and prostanoids have been found to mediate the parate class of purinoceptors (named the P2U-purinoceptor) dilator response to adenosine in some mammalian prepara- has been proposed where UTP > ATP (von Kigelgen et al., tions (Ciabattoni & Wennmalm, 1985; Vials & Burnstock, 1987; Saiag et al., 1990; 1992; Ralevic & Burnstock, 1991; 1993a). O'Connor, 1992). Lack of selective antagonists makes identi- In addition to activating receptors that are located on the fication of a separate P2U subclass difficult. Responses to UTP cell surface (Al-, A2- and A3-receptors), adenosine is thought are not affected by indomethacin (von Kilgelgen et al., 1987; to interact with an intracellular 'P-site' (Londos & Wolff, Ralevic & Burnstock, 1991; Vials & Burnstock, 1993b). 1977). Adenosine and 2-CA are found to activate this site In conclusion, this study has shown that adenosine induces which is believed to be linked to adenylyl cyclase (Londos et vasodilatation of the frog aorta by a novel PI-purinoceptor al., 1980). NECA and PIA are inactive at this site, which is also that has characteristics of the A3-receptor, the dilatation being insensitive to xanthine antagonism. It is unlikely that adeno- independent of the production of NO or a prostanoid. The sine is mediating vasodilatation via activation of an in- presence of a purinoceptor of the P2X family has been de- tracellular P-site, similar to that found in the guinea-pig aorta monstrated in the frog aorta: a,fJ-MeATP responses are an- (Collis & Brown, 1983), since 2-CA was inactive. tagonized by the action of suramin and PPADS, much of the The activity of ATP analogues on the frog aorta can be effect of ATP is via the production of a prostanoid, although characterized according to mammalian P2-purinoceptor clas- the receptor is novel in that it is not susceptible to desensiti- sification. ATP, a,fJ-MeATP, fI,y-MeATP and UTP all con- zation by a,fJ-MeATP. There is no evidence for the presence of centration-dependently constricted the frog aorta, ATP and a P2rpurinoceptor mediating vasodilatation in this prepara- a,fJ-MeATP being equipotent and more potent than UTP, fi,y- tion. MeATP or 2-MeSATP. The activity of a,f1-MeATP together with the more limited activity of 2-MeSATP suggest that the receptor is a member of the P2x-purinoceptor family. A P2- purinoceptor mediating vasoconstriction has also been de- scribed in the systemic vasculature of the trout (Wood, 1977). That the receptor is of the P2x subtype is supported further, since the contractile response to a,fi-MeATP is inhibited by the The authors are very grateful for the advice of Dr A.U. Ziganshin selective P2x-purinoceptor antagonist, PPADS (Lambrecht et during the preparation of this manuscript, and to Dr D. Christie for al., 1992; Ziganshin et al., 1993; 1994). This receptor differs invaluable editorial assistance. G.E. Knight & G. Burnstock Adenosine dilates and ATP constricts the frog aorta 877 References ABBRACCHIO, M.P. & BURNSTOCK, G. (1994). Purinoceptors: are DRURY, A.N. & SZENT-GYORGYI, A. (1929). The physiological there families of P2X and P2Y purinoceptors? Pharmacol. 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